【摘要】 目的:探讨蛋白激酶A（PKA）抑制剂H 89通过调控小鼠蛋白激酶Wee1B蛋白S15位点磷酸化状态影响小鼠1-细胞期受精卵的发育。方法:超排卵方法获得小鼠1-细胞期受精卵,经H-89预处理后显微注射Wee1B-mRNAs,相差显微镜观察受精卵的形态变化、计数卵裂率;放射自显影测定M期促进因子（MPF）活性;免疫印迹检测Wee1B蛋白表达、CDC2pTyr15和Wee1B-pSer15磷酸化及Wee1B-pSer15非磷酸化状态。结果:H-89持续存在时,在受精卵G₁和S期检测到Wee1B-Ser15的磷酸化条带,在G2期和M期检测到Wee1B-Ser15的非磷酸化条带;小鼠受精卵有丝分裂进程加快,但过表达Wee1B-WT和过表达突变体Wee1B-S15A/D均能延缓受精卵有丝分裂进程,逆转由H-89引起的促进分裂作用。结论:H-89抑制PKA活性,从而抑制Wee1B蛋白S15位点的磷酸化状态,但过表达Wee1B-S15A/D有效逆转H-89加速的G₂/M期转换,使受精卵的分裂延迟。提示Wee1B蛋白的S15位点可能是PKA作用的生理靶点。更多还原

【Abstract】 Objective:To study the role of N-[2-（p-bromocinnamylamino）ethyl]-5-isoquinolinesulfonamide·2HCl hydrate（H-89） in regulating the phosphorylation status of S15 site of WEE1 homolog 2（WeelB） on one-cell-stage mouse fertilized ovum.Methods:One-cell-stage mouse fertilized ovum were collected after superovulation,and were microinjected with or without TE buffer,WeelB-WT/KD mRNAs and WeelB-S15A/D mRNAs,respectively.Then they were cultured in M16 medium in the presence of H-89 and the protein expression level of WeelB was examined by Western blotting.The morphological changes and cleavage rate of mouse fertilized ovum were observed under phase-contrast microscope,and the M-phase promoting factor（MPF） activity of mouse fertilized ovum was detected by autoradiography and the phosphorylation status of CDC2-pTyr15 and Wee1B-pSer15 were observed by Western blotting.Results:The strong phosphorylated WeelB-Ser 15 band was observed at the G₁ and S phases,whereas no phosphorylation of WEE1B-Ser 15 was detected at G₂ and M phases in one-cell-stage fertilized ovum in the presence of H-89.Mitosis of mouse fertilized ovum was accelerated.However,WeelB-WT and WeelBS15A/D mutants delayed G₂/M transition induced by H-89.Conclusion:Protein kinase A（PKA） inhibition is responsible for the activation of MPF activity and can promote interphase in control and fertilized ovum injected with WeelB-KD mRNA.However,fertilized ovum microinjected with WeelB-WT and Wee1B-S15A/D mutants may delay G₂/M transition induced by H-89,suggesting residue S15 of WEE1 B is likely to be the direct target of PKA in mouse fertilized ovum.更多还原